Antimicrobial Resistance and Investigation of the Molecular Epidemiology of Listeria Monocytogenes in Dairy Products

A resistência antimicrobiana e investigação de epidemiologia molecular de Listeria monocytogenes em produtos lácteos ABSTracT Introduction: Listeria monocytogenes is a ubiquitous microorganism in nature and is responsible for listeriosis, an infectious disease caused by consumption of contaminated food. Methods: Molecular characterization was performed on 19 strains of Listeria monocytogenes (serovars 1/2a, 1/2b, 4b and 4c), isolated from dairy products in Rio Grande do Sul, Brazil. The molecular techniques applied were random amplification of polymorphic DNA and restriction enzyme analysis. In addition to the molecular analysis, the antimicrobial resistance profile was determined. Results: The strains studied showed a low degree of diversity. In relation to the antimicrobial resistance profile of those microorganisms from the samples analyzed, all of them were susceptible to the antimicrobials tested. conclusions: The molecular techniques that were used presented good discriminatory power for the strains studied. Furthermore, all of the samples that were analyzed were susceptible to the antimicrobials tested.

The genus Listeria is formed by six species, among which Listeria monocytogenes is known as one of the main pathogenic species in human beings.They consist of Grampositive rods that are capable of growing under adverse conditions such as low temperatures, acidic pH, high salt concentrations and the procedures that are applied by the food industry to halt the growth of pathogenic microorganisms.Due to their ubiquitous nature, these bacteria are constantly isolated from decomposing material, soil and water, and in this way, they can contaminate raw foods through cross-contamination 1 .
In general, most strains of L. monocytogenes isolated from clinical samples, food and the environment are susceptible to active antimicrobials against Gram-positive bacteria, and the treatment selected is a combination of ampicillin with an aminoglycoside, normally gentamicin 2 .Although all the 13 serovars of L. monocytogenes are capable of causing listeriosis, nearly 95% of the infection cases in humans are caused by 3 serotypes: 1/2a, 1/2b and 4b.In Brazil, one study has shown that serovar 1/2a is predominant in dairy products 3 .
The aim of the present study was to determine the genotype profile for L. monocytogenes that is predominant in southern Brazil, using molecular biology tools such as random amplification of polymorphic DNA (RAPD) and restriction endonuclease analysis (PCR-REA), on 19 strains isolated from dairy products.Furthermore, the antimicrobial resistance profile of these microorganisms against several antimicrobials that were tested was observed.

Bacterial strains and DNA extraction
The nineteen strains used in the present study were isolated from dairy products and were provided by the National Agricultural and Livestock-Rearing Laboratory in Porto Alegre (LANAGRO/RS).

Antimicrobial susceptibility test
All antimicrobial susceptibility tests were carried out using the standard disk diffusion method recommended by the NCCLS/CLSI guidelines, using ampicillin, gentamicin, vancomycin, ciprofloxacin, erythromycin, tetracycline, chloramphenicol and imipenem.None of the other antimicrobial agents tested had a standardized breakpoint for Listeria sp, so the breakpoints established for Staphylococcus sp were used 5 .
PcR-ReA analysis The procedure described was based on the method by Ericsson et al 6 , which consists of amplification of a 2,916bp fragment containing parts of the inlA and inlB genes, which have been correlated with virulence of L. monocytogenes, and on further cleavage of the DNA fragment using the restriction endonuclease AluI.PCR was performed using the primers FD (5'CGACAACATTTAGTGAACCGTG3') and FN (5' GCTGCTTTCGTCCAACCAATGAA 3'), synthesized by Invitrogen Brazil, Ltd.The samples were analyzed by means of gel electrophoresis in 2% agarose stained with ethidium bromide (0.5µg/ml).They were then viewed under UV transillumination and photographed using Kodak Digital Science TM DC120.

raPD analysis
The two primers used in this study (UBC127 and UBC155) were designed in accordance with Farber & Addison 7 .All PCR amplification was performed in a final volume of 25µl containing 1.5mM of MgCl 2 , 0.2mM of each dNTP, 1mM of each primer and 1.25U of Taq DNA polymerase.A thermal cycler (MJ Research, Inc.; PTC-100) was used for the PCR reaction.The cycling parameters used were: one cycle at 94°C for 2 min followed by 35 cycles at 94°C for 1 min, 35°C for 1 min and 72°C for 2.5 min, with a final extension of 5 min at 72°C.The PCR products were analyzed and photographed as described above.The reproduction of RAPD was assessed by carrying out least three independent trials.

Data analysis
The results obtained from the RAPD analysis were examined using the SPSS software.The similarity between isolates was calculated by means of the simple association coefficient and the grouping analysis by means of UPGMA (unweighted pair group method using average).The presence or absence of bands generated by RAPD was considered to be an alternative characteristic, and it was coded as 1 or 0, respectively.

Antibiotic susceptibility test
None of the 19 L. monocytogenes strains analyzed were resistant to the antimicrobials tested and only one of the isolates (A49) showed reduced susceptibility to ciprofloxacin antimicrobials.

PcR-ReA analysis
The DNA fragment of 2,916bp obtained from amplification of the genome of L. monocytogenes using the primers FD/FN that correspond the internal region of genes inlAB was digested using the restriction endonuclease AluI.The product from DNA cleavage made it possible to divide the strains analyzed into three different profiles.Most of the strains 8 presented a p1 profile including the control strains A51 and A56 8 .The p2 profile appeared in only one of the strains studied (A45) and the p3 profile was observed in the control strain ATCC7645 and strain A46 (Figure 1).

raPD analysis
The UBC 127 primer produced three to five DNA bands with a molecular size between 450bp and 2500bp (Figure 2).The profiles of the UBC 127 primer are shown in a dendrogram (Figure 3).The strains of L. monocytogenes that were studied could be divided into three main clusters, with a similarity level around 0.74.The first cluster included all the strains with serovars 1/2b, 4c and 4b, which were divided into three subgroups.The second one included only the two strains of L. monocytogenes isolated from dairy products with serovar 1/2a (A54 and A55).The third cluster separated the two control strains ATCC 7645 and A56, both with serovar 1/2a.It was also observed that the band for the highest molecular size (2.5Kb) was reproduced in all the isolates from dairy products except in strain A39, while the molecular band of 1.  in most of the strains of dairy products that were isolated, except in 1/2a serovars.The UBC 155 primer produced three to six DNA bands with molecular sizes of 250bp to 4,250bp (Figure 4).The profiles of the UBC 155 primer are shown in a dendrogram (Figure 5).The strains analyzed were divided into 3 main groups with a similarity level around 0.75.The first cluster involved all the strains of L. monocytogenes serovars 1/2b, 4b and 4c; this cluster could be further divided into four subgroups.The second group was formed by strains A54 and A55 of serovar 1/2a, isolated from dairy products in southern Brazil, and strain A56 isolated in Canada.ATCC 7,645 represented the third cluster.Listeriosis is an important and severe disease caused by the L. monocytogenes microorganism, in which the main source of disease transmission is ingestion of contaminated food, especially dairy products, which have been responsible for outbreaks of listeriosis 1 .In Brazil, there is a lack of information on listeriosis, particularly because there are no official statistics about the disease, given that its notification is not compulsory.There is no reporting of outbreaks; rather, only isolated cases are noted 9 .
The treatment chosen for listeriosis is the administration of penicillin or ampicillin associated with an aminoglycoside, usually gentamicin.For patients who are allergic to penicillin, the treatment can be administered successfully using a combination of vancomycin and an aminoglycoside or sulfamethoxazole-trimethoprim (TMP-SMX), in association with rifampicin.The use of cephalosporin is not suggested because strains of L. monocytogenes are generally resistant to those antimicrobials 10 .The incidence of resistance among isolates from clinical samples and food continues to be low.However, the emergence of resistant strains is being described in many studies 11 12 .In 1988, the first strain of multi-resistant L. monocytogenes was isolated from human clinical material in France.The resistance was the result of plasmidial transfer, which was believed to have originated from , , ,

DISCUSSIONFIGuRe 5 -FIGuRe 2 -
FIGuRe 5 -Dendrogram of Listeria monocytogenes raPD for strains isolated in southern Brazil and strains A51, A55 and ATcc 7,645, using the primer uBc 155 and serovar discrimination.

3 -Dendrogram of Listeria monocytogenes raPD for strains isolated in southern Brazil and strains A51, A56 and ATcc 7,645, using the primer uBc127 and serovar discrimination.
ATCCFIGuRe